结核分枝杆菌MurA的优化表达及功能分析
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摘要
结核分枝杆菌(Mycobacterium tuberculosis)是引起结核病的病原体。从世界范围来看,结核病仍是一种发病率和死亡率极高的传染性疾病,全球人口的三分之一感染了结核分枝杆菌,而且,其发病率也呈逐年上升的趋势。其主要原因之一是由于耐多药(multi drug resistant, MDR)甚至是广泛耐药(extensive drug resistant, XDR)结核杆菌的出现,许多一线甚至一些二线抗结核药物都无法有效治疗结核病。因此,当前的首要任务是从结核分枝杆菌自身寻找新的靶点,以此来研发新的抗结核药物。
分枝杆菌细胞壁的核心结构由分枝菌酸,聚阿拉伯半乳糖和肽聚糖构成。其中,肽聚糖是维持细菌细胞形态和渗透压平衡的机构基础,其组成及结构可能与G-细菌肽聚糖相似,即由N-乙酰葡糖胺和N-乙酰胞壁酸交替连接形成的多糖链与短肽链交叉连接形成网状大分子机构。UDP-乙酰胞壁酸是N-乙酰胞壁酸的糖基供体,两个酶催化由UDP-乙酰葡糖胺转变为UDP-乙酰胞壁酸的代谢途径。MurA (UDP-N-乙酰葡糖胺1-羧基乙烯基转移酶)是催化此条途径的第一个关键酶,由于此反应途径无代谢旁路,并且在哺乳动物中不存在此代谢途径,因此,催化此代谢途径的酶可能是抗结核药物的作用靶点。
为了建立体外筛选抗结核药物的分子模型,我们首先要克隆编码结核分枝杆菌UDP-N-乙酰葡糖胺1-羧基乙烯基转移酶的murA基因,然后表达Tb MurA蛋白,为进一步研究UDP-N-乙酰葡糖胺1-羧基乙烯基转移酶的功能及建立筛选抗结核药物的酶反应体系提供物质保障。
本论文的目的：(1)用pET29b表达载体在大肠杆菌BL21(DE3)中表达结核分枝杆菌MurA蛋白,优化表达条件以期获得可溶性蛋白；(2)分离、溶解、纯化及鉴定包含体形式的Tb MurA蛋白,并用纯化的Tb MurA蛋白免疫兔子以制备多克隆抗体,对抗体的效价和特异性进行检测；(3)用pVV 16表达载体在耻垢分枝杆菌mc2155中表达Tb MurA蛋白,并用亲和层析技术纯化Tb MurA蛋白,用SDS-PAGE以及Western blotting鉴定所纯化的Tb MurA蛋白；(4)用高效液相色谱(HPLC)法和化学显色法两种方法测定MurA蛋白的酶活性。
本论文获得的结果：
1.表达载体pET29b-Tb murA的构建
用NdeⅠ和HindⅢ双酶切pMD18-Tb murA质粒,回收和纯化TbmurA基因,将其连接到pET29b表达载体的NdeⅠ和HindⅢ位点,构建pET29b-Tb murA表达质粒。用SmaⅠ酶切的方法鉴定阳性重组质粒。MurA蛋白的C端与pET29b表达载体上的6个连续的组氨酸标签形成融合蛋白,便于目的蛋白的检测和纯化。
2. Tb MurA蛋白在大肠杆菌BL21(DE3)中的诱导表达及表达条件的优化
将pET29b-Tb murA质粒转化到BL21(DE3)感受态细胞中,诱导携带pET29b-Tb murA质粒的BL21(DE3)表达重组蛋白。用超声方法破碎诱导后的BL21(DE3),对上清和沉淀组分进行SDS-PAGE和Western blotting分析,结果表明Tb MurA蛋白在BL21(DE3)中形成包含体。优化表达条件,通过降低IPTG的浓度、诱导温度及与分子伴侣共表达等,但没有改善目的蛋白的溶解性。
3.包含体的分离、溶解、纯化及鉴定
将包含体进行了分离,用溶解缓冲液溶解,获得了很好的溶解效果,获得了大量的可溶性蛋白。用组氨酸-Ni2+亲和层析技术纯化溶解后的Tb MurA蛋白。对纯化的蛋白进行蛋白质定量(考马斯亮蓝法),SDS-PAGE和Western blotting分析结果表明我们获得了纯Tb MurA蛋白。
4.多克隆抗体的制备及效价、特异性检测
将纯化的Tb MurA蛋白溶液制备成免疫用抗原,免疫家兔,制备抗血清,通过间接法酶联免疫吸附试验测得抗血清的抗体滴度为1：78125,采用抗血清及偶联碱性磷酸酶的山羊抗兔IgG二抗分别对在耻垢分枝杆菌中表达的TbMurA蛋白和野生耻垢分枝杆菌上清蛋白进行Western Blotting分析,结果表明制备的MurA多克隆抗体特异性较好。
5.表达载体pVV16-Tb murA的构建
用NdeⅠ和HindⅢ双酶切pMD18-Tb murA质粒,回收和纯化TbmurA基因,将其连接到pVV1 6表达载体的NdeⅠ和HindⅢ位点,构建pVV16-Tb murA表达质粒。用XhoI酶切的方法鉴定阳性重组质粒。TbMurA蛋白的C端与pVV1 6表达载体上的组氨酸标签形成融合蛋白,便于目的蛋白的检测和纯化。
6. Tb MurA蛋白在耻垢分枝杆菌mc2155中的表达
将pVV16-Tb murA质粒转化到mc21 55感受态细胞中,37℃震荡培养48小时表达目的蛋白,用超声方法破碎培养后的细菌,对上清和沉淀组分进行SDS-PAGE和Western blotting分析,结果表明Tb MurA蛋白在mc2155中可溶性表达。
7.采用亲和层析技术纯化Tb MurA蛋白
pVV16表达载体使重组MurA蛋白C端带有组氨酸标签,因此,用组氨酸-Ni2+亲和层析技术纯化Tb MurA蛋白。对纯化的蛋白进行蛋白质定量(考马斯亮蓝法),SDS-PAGE和Western blotting分析结果表明我们获得了纯Tb MurA蛋白。
8.建立Tb MurA蛋白的酶活性测定方法
(1)高效液相色谱法：采用Nova-Pak C18(3.9×150mm,4μm)色谱柱,以20 mM三乙胺-醋酸缓冲液(pH 4.0)为流动相,流速0.5mLmin-1,在波长260nm处检测反应底物UDP-GlcNAc的减少,其洗脱时间为6.3min。
(2)化学显色法：反应产物之一为磷酸,磷酸与钼酸铵形成磷钼酸复合物后使孔雀石绿颜色由黄绿变为蓝绿,用酶标仪在630 nm处检测吸光值变化,以测定所生成磷酸的含量。
结论：在本研究中,我们构建了pET29b-Tb murA表达载体,在大肠杆菌BL21(DE3)中表达出大量以包含体性形式存在的Tb MurA蛋白。
我们用包含体中的Tb MurA蛋白制备了多克隆抗体,分别用ELISA和Western blotting进行检测和鉴定,多克隆抗体有较高的效价和特异性。
我们构建了pVV16-Tb murA表达载体,在耻垢分枝杆菌mc2155中表达出以可溶性形式存在的Tb MurA蛋白,并鉴定了Tb MurA蛋白的酶活性。
Mycobacterium tuberculosis is the pathogen that causes tuberculosis. As a global issue, tuberculosis (TB) is still an infectious disease with extremely high morbidity and mortality, catched by nearly one thirds of worldwide populations, moreover the incidence is rising yearly. Major problem is the emergence of multi-drug and extra-drug resistant strains makes invalid of many anti-TB drugs. Therefore, identifying new targets from M. tuberculosis itself and developing new anti-tuberculosis drugs may become a chief task currently.
The core of the mycobacterial cell wall consists of mycolic acid, arabinogalactan, and peptidoglycan for connection. One of the best known and most validated targets for antibacterial therapy is the machinery for peptidoglycan biosynthesis. The biosynthetic pathway of Mycobacterium tuberculosis peptidoglycan is a complex two-stage process. The first stage, which occurs in the cytoplasm, is the formation of the monomeric building block N-acetylglucosamine-N-acetylmuramyl pentapeptide. The first committed step in the pathway is the transfer of an enolpyruvate residue from phosphoenolpyruvate (PEP) to position 3 of UDP-N-acetylglu-cosamine. This reaction is catalysed by the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA). MurA is ubiquitous to both Gram-positive and Gram-negative bacteria, and has no homologue in mammalian cells. Therefore, MurA is a potential target to develop anti-TB drugs.
Our long-term objective is to establish a molecule model of screening anti-TB drug. Therefore, we must first clone the gene murA which encodes the enzyme MurA, and then express soluble MurA protein. Soluble MurA will be utilized for further purification and study the kinetics of MurA as well as development of enzyme assay to screen anti-Tb drugs.
The objectives of this study:(1) to express M. tuberculosis MurA protein by pET29b express vector in E.coli BL21(DE3) and optimize the expression conditions in order to obtain the soluble MurA; (2) to separate and solubolize inclusion body-forming MurA protein and purify MurA protein by affinity chromatography. To identify the purified MurA protein by SDS-PAGE and Western blotting and prepare anti-MurA polyclonal antibody by injecting rabbit with purified MurA protein and detect the serum specificity and its titer; (3) to express M. tuberculosis MurA protein by pVV16 express vector in M.smegmatis mc2155; (4) to establish methods to detect the activity of MurA enzyme.
The results are followings:
1. Expression vector pET29b-Tb murA was constructed.
pMD18-Tb murA was digested by NdeⅠand HindⅢ, then Tb murA gene fragment was purified and ligated into the NdeⅠand HindⅢsites of vector pET29b to generate pET29b-Tb murA. The C-terminus of MurA protein was fused with histidine tag in pET29b vector.
2. Tb MurA protein was expressed in E.coli BL21(DE3) and the expression conditions was optimized.
pET29b-Tb murA were transformed into BL21(DE3) competent cells. BL21(DE3) cells carring pET29b-Tb murA were induced. Induced BL21(DE3) cells were sonicated and both supernatant and pellet fractions were analyzed by SDS-PAGE and Western blotting. MurA protein was expressed in E.coli BL21(DE3),but they were insoluble in inclusion body. Optimize the expression conditions, for example, to reduse the concen-tration of IPTG, to induce under lower temperature condition and to coexpress with chaperons, and so on. The Tb MurA still formed inclusion bodies.
3. Inclusion body-forming Tb MurA protein was separated, solubolized, purified and detected.
The inclusion body-forming Tb MurA protein was separated, solubolized with different buffers and obtained lots of soluble Tb MurA protein. Tb MurA protein was purified by histidine-Ni2+ affinity chromatography. The elution fraction was quantified by coomassie brilliant blue method. The purified MurA protein was confirmed by SDS-PAGE and Western blotting.
4. Anti-Tb MurA polyclonal antibody was prepared and identified.
Purified Tb MurA was prepared with particular means for injecting rabbit. Antiserum was separated and assayed for antibody titer as 1:78125 by enzyme-linked immunosorbent assay. Tb MurA protein expressed in mc2155 and protein of M. smegmatis mc2155 was detected by antiserum followed by antirabbit-IgG-conjugated with alkaline phosphatase. The result showed anti-MurA polyclonal antibody had high specificity.
5. Expression vector pVV16-Tb murA was constructed.
pMD18-Tb murA was digested by NdeⅠand HindⅢ, then murA gene fragment was purified and ligated into the NdeⅠand HindⅢsites of vector pVV16 to generate pVV16-Tb murA. The C-terminus of Tb MurA protein was fused with histidine tag in pVV16 vector.
6. Tb MurA protein in M. smegmatis mc2155 was expressed and identified.
pVV16-Tb murA was transformed into M. smegmatis mc2155 competent cells to express the Tb MurA protein. The mc2155 cells were broken by sonication, and the proteins from both supernatant and pellet fractions were analyzed by SDS-PAGE and Western blotting. The results showed that Tb MurA protein in mc2155 was soluble expressed.
7. Tb MurA protein was purified by affinity chromatography.
The C-terminus of Tb MurA was fused with histidine tag in pVV16, therefore Tb MurA was purified by histidine-Ni2+ affinity chromatography. The elution fraction was quantified by coomassie brilliant blue method. The purified Tb MurA protein was confirmed by SDS-PAGE and Western blotting.
8. The enzyme assay of Tb MurA was established.
(1) HPLC:UDP-G1cNAc was separated with Nova-Pak C18 at a flow rate of 0.5mLmin-1 under 20 mM triethylamine-acetic acid buffer (pH 4.0) and was monitored at 260 nm. The peak of UDP-G1cNAc was appeared at 6.3 min approximately. Its reduction indicated that MurA has activity.
(2) Chemical colorimetric assays:The inorganic phosphate produced in the reation was detected. The reaction of inorganic phosphate and ammonium molybdate results in the formation of molybdenum blue, which turns malachite green from yellow to blue. The OD value at 630 nm was detected.
Conclusions:
In this study, we constructed pET29b-Tb murA expression vector and overexpressed recombinant Tb MurA protein in inclusion body and anti-MurA polyclonal antibody was prepared.
We constructed pVV16-Tb murA expression vector and expressed considerable soluble recombinant Tb MurA protein in M. smegmatis mc2155.
We established Tb MurA enzyme assay and identified Tb MurA enzyme activity.
分枝杆菌细胞壁的核心结构由分枝菌酸,聚阿拉伯半乳糖和肽聚糖构成。其中,肽聚糖是维持细菌细胞形态和渗透压平衡的机构基础,其组成及结构可能与G-细菌肽聚糖相似,即由N-乙酰葡糖胺和N-乙酰胞壁酸交替连接形成的多糖链与短肽链交叉连接形成网状大分子机构。UDP-乙酰胞壁酸是N-乙酰胞壁酸的糖基供体,两个酶催化由UDP-乙酰葡糖胺转变为UDP-乙酰胞壁酸的代谢途径。MurA (UDP-N-乙酰葡糖胺1-羧基乙烯基转移酶)是催化此条途径的第一个关键酶,由于此反应途径无代谢旁路,并且在哺乳动物中不存在此代谢途径,因此,催化此代谢途径的酶可能是抗结核药物的作用靶点。
为了建立体外筛选抗结核药物的分子模型,我们首先要克隆编码结核分枝杆菌UDP-N-乙酰葡糖胺1-羧基乙烯基转移酶的murA基因,然后表达Tb MurA蛋白,为进一步研究UDP-N-乙酰葡糖胺1-羧基乙烯基转移酶的功能及建立筛选抗结核药物的酶反应体系提供物质保障。
本论文的目的：(1)用pET29b表达载体在大肠杆菌BL21(DE3)中表达结核分枝杆菌MurA蛋白,优化表达条件以期获得可溶性蛋白；(2)分离、溶解、纯化及鉴定包含体形式的Tb MurA蛋白,并用纯化的Tb MurA蛋白免疫兔子以制备多克隆抗体,对抗体的效价和特异性进行检测；(3)用pVV 16表达载体在耻垢分枝杆菌mc2155中表达Tb MurA蛋白,并用亲和层析技术纯化Tb MurA蛋白,用SDS-PAGE以及Western blotting鉴定所纯化的Tb MurA蛋白；(4)用高效液相色谱(HPLC)法和化学显色法两种方法测定MurA蛋白的酶活性。
本论文获得的结果：
1.表达载体pET29b-Tb murA的构建
用NdeⅠ和HindⅢ双酶切pMD18-Tb murA质粒,回收和纯化TbmurA基因,将其连接到pET29b表达载体的NdeⅠ和HindⅢ位点,构建pET29b-Tb murA表达质粒。用SmaⅠ酶切的方法鉴定阳性重组质粒。MurA蛋白的C端与pET29b表达载体上的6个连续的组氨酸标签形成融合蛋白,便于目的蛋白的检测和纯化。
2. Tb MurA蛋白在大肠杆菌BL21(DE3)中的诱导表达及表达条件的优化
将pET29b-Tb murA质粒转化到BL21(DE3)感受态细胞中,诱导携带pET29b-Tb murA质粒的BL21(DE3)表达重组蛋白。用超声方法破碎诱导后的BL21(DE3),对上清和沉淀组分进行SDS-PAGE和Western blotting分析,结果表明Tb MurA蛋白在BL21(DE3)中形成包含体。优化表达条件,通过降低IPTG的浓度、诱导温度及与分子伴侣共表达等,但没有改善目的蛋白的溶解性。
3.包含体的分离、溶解、纯化及鉴定
将包含体进行了分离,用溶解缓冲液溶解,获得了很好的溶解效果,获得了大量的可溶性蛋白。用组氨酸-Ni2+亲和层析技术纯化溶解后的Tb MurA蛋白。对纯化的蛋白进行蛋白质定量(考马斯亮蓝法),SDS-PAGE和Western blotting分析结果表明我们获得了纯Tb MurA蛋白。
4.多克隆抗体的制备及效价、特异性检测
将纯化的Tb MurA蛋白溶液制备成免疫用抗原,免疫家兔,制备抗血清,通过间接法酶联免疫吸附试验测得抗血清的抗体滴度为1：78125,采用抗血清及偶联碱性磷酸酶的山羊抗兔IgG二抗分别对在耻垢分枝杆菌中表达的TbMurA蛋白和野生耻垢分枝杆菌上清蛋白进行Western Blotting分析,结果表明制备的MurA多克隆抗体特异性较好。
5.表达载体pVV16-Tb murA的构建
用NdeⅠ和HindⅢ双酶切pMD18-Tb murA质粒,回收和纯化TbmurA基因,将其连接到pVV1 6表达载体的NdeⅠ和HindⅢ位点,构建pVV16-Tb murA表达质粒。用XhoI酶切的方法鉴定阳性重组质粒。TbMurA蛋白的C端与pVV1 6表达载体上的组氨酸标签形成融合蛋白,便于目的蛋白的检测和纯化。
6. Tb MurA蛋白在耻垢分枝杆菌mc2155中的表达
将pVV16-Tb murA质粒转化到mc21 55感受态细胞中,37℃震荡培养48小时表达目的蛋白,用超声方法破碎培养后的细菌,对上清和沉淀组分进行SDS-PAGE和Western blotting分析,结果表明Tb MurA蛋白在mc2155中可溶性表达。
7.采用亲和层析技术纯化Tb MurA蛋白
pVV16表达载体使重组MurA蛋白C端带有组氨酸标签,因此,用组氨酸-Ni2+亲和层析技术纯化Tb MurA蛋白。对纯化的蛋白进行蛋白质定量(考马斯亮蓝法),SDS-PAGE和Western blotting分析结果表明我们获得了纯Tb MurA蛋白。
8.建立Tb MurA蛋白的酶活性测定方法
(1)高效液相色谱法：采用Nova-Pak C18(3.9×150mm,4μm)色谱柱,以20 mM三乙胺-醋酸缓冲液(pH 4.0)为流动相,流速0.5mLmin-1,在波长260nm处检测反应底物UDP-GlcNAc的减少,其洗脱时间为6.3min。
(2)化学显色法：反应产物之一为磷酸,磷酸与钼酸铵形成磷钼酸复合物后使孔雀石绿颜色由黄绿变为蓝绿,用酶标仪在630 nm处检测吸光值变化,以测定所生成磷酸的含量。
结论：在本研究中,我们构建了pET29b-Tb murA表达载体,在大肠杆菌BL21(DE3)中表达出大量以包含体性形式存在的Tb MurA蛋白。
我们用包含体中的Tb MurA蛋白制备了多克隆抗体,分别用ELISA和Western blotting进行检测和鉴定,多克隆抗体有较高的效价和特异性。
我们构建了pVV16-Tb murA表达载体,在耻垢分枝杆菌mc2155中表达出以可溶性形式存在的Tb MurA蛋白,并鉴定了Tb MurA蛋白的酶活性。
Mycobacterium tuberculosis is the pathogen that causes tuberculosis. As a global issue, tuberculosis (TB) is still an infectious disease with extremely high morbidity and mortality, catched by nearly one thirds of worldwide populations, moreover the incidence is rising yearly. Major problem is the emergence of multi-drug and extra-drug resistant strains makes invalid of many anti-TB drugs. Therefore, identifying new targets from M. tuberculosis itself and developing new anti-tuberculosis drugs may become a chief task currently.
The core of the mycobacterial cell wall consists of mycolic acid, arabinogalactan, and peptidoglycan for connection. One of the best known and most validated targets for antibacterial therapy is the machinery for peptidoglycan biosynthesis. The biosynthetic pathway of Mycobacterium tuberculosis peptidoglycan is a complex two-stage process. The first stage, which occurs in the cytoplasm, is the formation of the monomeric building block N-acetylglucosamine-N-acetylmuramyl pentapeptide. The first committed step in the pathway is the transfer of an enolpyruvate residue from phosphoenolpyruvate (PEP) to position 3 of UDP-N-acetylglu-cosamine. This reaction is catalysed by the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA). MurA is ubiquitous to both Gram-positive and Gram-negative bacteria, and has no homologue in mammalian cells. Therefore, MurA is a potential target to develop anti-TB drugs.
Our long-term objective is to establish a molecule model of screening anti-TB drug. Therefore, we must first clone the gene murA which encodes the enzyme MurA, and then express soluble MurA protein. Soluble MurA will be utilized for further purification and study the kinetics of MurA as well as development of enzyme assay to screen anti-Tb drugs.
The objectives of this study:(1) to express M. tuberculosis MurA protein by pET29b express vector in E.coli BL21(DE3) and optimize the expression conditions in order to obtain the soluble MurA; (2) to separate and solubolize inclusion body-forming MurA protein and purify MurA protein by affinity chromatography. To identify the purified MurA protein by SDS-PAGE and Western blotting and prepare anti-MurA polyclonal antibody by injecting rabbit with purified MurA protein and detect the serum specificity and its titer; (3) to express M. tuberculosis MurA protein by pVV16 express vector in M.smegmatis mc2155; (4) to establish methods to detect the activity of MurA enzyme.
The results are followings:
1. Expression vector pET29b-Tb murA was constructed.
pMD18-Tb murA was digested by NdeⅠand HindⅢ, then Tb murA gene fragment was purified and ligated into the NdeⅠand HindⅢsites of vector pET29b to generate pET29b-Tb murA. The C-terminus of MurA protein was fused with histidine tag in pET29b vector.
2. Tb MurA protein was expressed in E.coli BL21(DE3) and the expression conditions was optimized.
pET29b-Tb murA were transformed into BL21(DE3) competent cells. BL21(DE3) cells carring pET29b-Tb murA were induced. Induced BL21(DE3) cells were sonicated and both supernatant and pellet fractions were analyzed by SDS-PAGE and Western blotting. MurA protein was expressed in E.coli BL21(DE3),but they were insoluble in inclusion body. Optimize the expression conditions, for example, to reduse the concen-tration of IPTG, to induce under lower temperature condition and to coexpress with chaperons, and so on. The Tb MurA still formed inclusion bodies.
3. Inclusion body-forming Tb MurA protein was separated, solubolized, purified and detected.
The inclusion body-forming Tb MurA protein was separated, solubolized with different buffers and obtained lots of soluble Tb MurA protein. Tb MurA protein was purified by histidine-Ni2+ affinity chromatography. The elution fraction was quantified by coomassie brilliant blue method. The purified MurA protein was confirmed by SDS-PAGE and Western blotting.
4. Anti-Tb MurA polyclonal antibody was prepared and identified.
Purified Tb MurA was prepared with particular means for injecting rabbit. Antiserum was separated and assayed for antibody titer as 1:78125 by enzyme-linked immunosorbent assay. Tb MurA protein expressed in mc2155 and protein of M. smegmatis mc2155 was detected by antiserum followed by antirabbit-IgG-conjugated with alkaline phosphatase. The result showed anti-MurA polyclonal antibody had high specificity.
5. Expression vector pVV16-Tb murA was constructed.
pMD18-Tb murA was digested by NdeⅠand HindⅢ, then murA gene fragment was purified and ligated into the NdeⅠand HindⅢsites of vector pVV16 to generate pVV16-Tb murA. The C-terminus of Tb MurA protein was fused with histidine tag in pVV16 vector.
6. Tb MurA protein in M. smegmatis mc2155 was expressed and identified.
pVV16-Tb murA was transformed into M. smegmatis mc2155 competent cells to express the Tb MurA protein. The mc2155 cells were broken by sonication, and the proteins from both supernatant and pellet fractions were analyzed by SDS-PAGE and Western blotting. The results showed that Tb MurA protein in mc2155 was soluble expressed.
7. Tb MurA protein was purified by affinity chromatography.
The C-terminus of Tb MurA was fused with histidine tag in pVV16, therefore Tb MurA was purified by histidine-Ni2+ affinity chromatography. The elution fraction was quantified by coomassie brilliant blue method. The purified Tb MurA protein was confirmed by SDS-PAGE and Western blotting.
8. The enzyme assay of Tb MurA was established.
(1) HPLC:UDP-G1cNAc was separated with Nova-Pak C18 at a flow rate of 0.5mLmin-1 under 20 mM triethylamine-acetic acid buffer (pH 4.0) and was monitored at 260 nm. The peak of UDP-G1cNAc was appeared at 6.3 min approximately. Its reduction indicated that MurA has activity.
(2) Chemical colorimetric assays:The inorganic phosphate produced in the reation was detected. The reaction of inorganic phosphate and ammonium molybdate results in the formation of molybdenum blue, which turns malachite green from yellow to blue. The OD value at 630 nm was detected.
Conclusions:
In this study, we constructed pET29b-Tb murA expression vector and overexpressed recombinant Tb MurA protein in inclusion body and anti-MurA polyclonal antibody was prepared.
We constructed pVV16-Tb murA expression vector and expressed considerable soluble recombinant Tb MurA protein in M. smegmatis mc2155.
We established Tb MurA enzyme assay and identified Tb MurA enzyme activity.
引文
1. Vollmer W, Blanot D.Biosynthesis of the bacterial peptidoglycan unit. New Comprehensive Biochemistry 2008; 7:149-154.
2. 陈国胜,谷欣,张想竹.细菌肽聚糖及其应用.安阳工学院学报2007;6：36-38.
3. 和致中.细菌细胞壁肽聚糖的分类.微生物学通报1992；19(3)：174-179.
4. Green DW. The bacterial cell wall as a source of antibacterial target. Expert Opin.Ther.Tar 2002; 6:1-19.
5. Walsh CT. Enzymes in the D-alanine branch of bacterial cell wall peptidoglycan assembly. J.Biol.Chem 1989; 264:2393-2396.
6. Mao Y, Zhiwen Zhang. Molecular cloning and expression analysis of peptidoglycan recognition protein Ⅱ gene of the large yellow croaker.J.Biotec 2008; 7:1278-1283.
7. Yukari F, Yasuko K. Synthesis of crosslinked peptidoglycan fragments for investigation of their immunobiological functions.Tetrahedron Letters 2009; 4:13-18.
8. Koch AL. Bacterial wall as target for attack:past, present,an future research.Clin.Microbiol Rev 2003;16:673-687.
9. Jenny BS, Brian G. An amino acid substitution that blocks the deacylation step in the enzyme mechanism of penicillin-binding protein 5 of Escherichia coli. Federation of European Biochemical Societies.19841; 25-31.
10. Clifton E, Dean C, Michael R. Targeting the Formation of the Cell Wall Core of M. tuberculosis. Infectious Disorders-Drug Targets 2007; 7:182-202.
II. Ahmed E, Francois S, Roger C. Structure and function of the Mur enzymes: development of novel inhibitors.MolecularMicrobiology 2003; 47(1):1-12.
12. Eschenburg S, Schonbrunn E. Comparative Xray analysis of the un-liganded fosfomycin-target murA.Proteins 2000; 40:290-298.
13. Marquardt JL, Brown ED, Walsh C., Anderson KS. Isolation and structural elucidation of tetrahedral intermediate in the UDP-N-acetylglucosamine enolpyruvoyl transferase enzymatic pathway. J Am Chem Soc 1993; 115:10398.
14. Skarzynski T, Kim DH, Lees WJ, Walsh CT, Duncan K. Stereochemical course of enzymatic enolpyruvyl transfer and catalytic conformation of the active site revealed by the crystal structure of the fluorinated analogue of the reaction tetrahedral intermediate bound to the active site of the C115A mutant of MurA.Biochemistry 1998; 37:2572-2577.
15. Brown ED, Marquardt JL, Lee JP, Walsh CT, Anderson KS. Detection and characterization of a phospholactoyl-enzyme adduct in the reaction catalyzed by UDP-N-acetylglucosamine enolpyruvoyl transferase, MurZ. Biochemistry 1994; 33:10638-10645.
16. Kim DH, Lees WJ, Kempsell KE, Lane WS, Duncan K, Walsh CT. Characterization of a Cys 115 to Asp substitution in the Escherichia coli cell wall biosynthetic enzyme UDP-G1cNAc enolpyruvyl transferase (MurA) that confers resistance to inactivation by the antibiotic fosfomycin. Biochemistry 1996; 35: 4923-4928.
17. Ramilo C, Appleyard RJ, Wanke C, Krekel F, Amrhein N, Evans JN. Detection of the covalent intermediate of UDP-N-acetylglucosamine enolpyruvyl transferase by solution-state and time-resolved solid-state NMR spectroscopy. Biochemistry 1994; 33:15071-15079.
18. Schonbrunn E, Svergun DI, Amrhein N, Koch MH.Studies on the conformational changes in the bacterial cell wall biosynthetic enzyme UDP-N-acetylglucosamine enolpyruvyltransferase (MurA). Eur J Biochem 1998; 253:406-412.
19. Samland AK, Amrhein N, Macheroux P. Lysine 22 in UDP-N-acetylglucosamine enolpyruvyl transferase from Enterobacter cloacae is crucial for enzymatic activity and the formation of covalent adducts with the substrate phosphor-enolpyruvate and the antibiotic fosfomycin.Biochemistry 1999;38:13162-13169.
20. Du W, Brown JR, Sylvester DR, Huang J, Chalker AF. Two active forms of UDP-Nacetylglucosamine enolpyruvyl transferase in grampositive bacteria. J Bacteriol 2000; 182:4146-4152.
21. Nicolle LE. Urinary tract infection:traditional pharmacologic therapies. Am J Med 2002; 113 (Suppl.1A):35-44.
22. Marquardt JL, Brown ED, Lane WS, Haley TM,lchikawa Y, Wong CH, Walsh CT. Kinetics,stoichiometry, and identification of the reactive thiolate in the inactivation of UDP-G1cNAc enolpyruvoyl transferase by the antibiotic fosfomycin. Biochemistry 1994; 33:10646-10651.
23. Kahan FM, Kahan JS, Cassidy PJ, Kropp H.The mechanism of action of fosfomycin (phosphonomycin).Ann NY Acad Sci 1974;235:364-386.
24. Marquardt J., Siegele DA, Kolter R, Walsh CT.Cloning and sequencing of Escherichia coli murZ and purification of its product, a UDP-N- acetylglucosamine enolpyruvyl transferase. J Bacteriol 1992; 174:5748-5752.
25. O'Hara K. Two different types of fosfomycin resistance in clinical isolates of Klebsiella pneumoniae. FEMS Microbiol Lett 1993; 114:9-16.
26. Arca P, Reguera G, Hardisson C. Plasmidencoded fosfomycin resistance in bacteria isolated from the urinary tract in a multicentre survey. J Antimicrob Chemother 1997; 40:393-399.
27. Horii T, Kimura T, Sato K, Shibayama K, Ohta M.Emergence of fosfomycin-resistant isolates of Shiga-like toxin-producing Escherichia coli 026. Antimicrob Agents Chemother 1997; 43:789-793.
28. Baum EZ, Montenegro DA, Licata L, Turchi I, Webb GC, Foleno BD, Bush K. Identification and characterization of new inhibitors of the Escherichia coli MurA enzyme. Antimicrob Agents Chemother 2001; 45:3182-3188.
29. Schonbrunn E, Eschenburg S, Krekel F, Luger K, Amrhein N. Role of the loop containing residue 115 in the induced-fit mechanism of the bacterial cell wall biosynthetic enzyme MurA. Biochemistry 2000; 39:2164-2173.
30. Schonbrunn E, Svergun DI, Amrhein N, Koch MH.Studies on the conformational changes in the bacterial cell wall biosynthetic enzyme UDP-N-acetylglucosamine enolpyruvyltransferase (MurA). Eur J Biochem 1998; 253:406-412.
31. De Smet KA, Kempsell KE, Gallagher A, Duncan K, Young, DB. Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis. Microbiology 1999;145:3177-3184
32. Jose'Molina-Lo'pez, Franc,ois Sanschagrin, Roger C. Levesque. A peptide inhibitor of MurA UDP-N-acetylglucosamine enolpyruvyl transferase:The first committed step in peptidoglycan biosynthesis. Peptides 2006; 27:3115-3121.
1. World Health Organization. Global tuberculosis control:Surveillance, Planning, Financing. WHO Report 2005. Geneva, Switzerland, WHO/HTM/TB/2005.349.
2. Murray CJ, Jopez AD.Global mortality, disability, and the contribution of risk factors:Global Burden of Disease Survey.Lancet 1997; 349(9063):1436-1442.
3. Dye C, Scheele S,Dolin P.Global burden of tuberculosis estimated incidence, prevalence and mortality by country.JAMA 1999; 282(7):677-686.
4. Susan ED, Richard EC. From magic bullets back to the Magic Mountain:the rise of extensively drug-resistant tuberculosis. Nature Medicine.2007; 13(3):295-8.
5. Raviglione MC, Smith IM. XDR Tuberculosis-Implications for Global Public Health. N. Engl. J. Med.2007; 356:656-659.
6. Wise J. Southern Africa is moving swiftly to combat the threat of XDR-TB. Bull World Health Organ.2006; 84(12):924-5.
7. Brennan PJ, Nikaido H. The envelope of mycobacteria. Annual Reviews of Biochemistry 1995; 64:29-63.
8. Crick DC, Brennan PJ, McNeil MR. The cell wall of Mycobacterium tuberculosis. In Tuberculosis (2 nd Edition) 2004; 115-134.
9. 赵乃昕,高屹.应重视肽聚糖结构在细菌分类中的意义-从某些教科书的内容欠妥论述谈起.微生物学通报2004；31(3)：174-175.
10. Ahmed El, Francois Sanschagrin, Roger C. Structure and function of the Mur enzymes: development of novel inhibitors, Molecular Microbiology 2003; 47 (1):1-12.
11. Gordon E, Flouret B, Chantalat L, Heijenoort J, Mengin-Lecreulx D, Dideberg O. Crystal structure of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase from Escherichia coli,J Biol Chem 2001;276 (10):10999-11006.
12. Jose'Molina-Lo'pez, Francois Sanschagrin, Roger C. A peptide inhibitor of MurA UDP-N-acetylglucosamine enolpyruvyl transferase:The first committed step in peptidoglycan biosynthesis, Peptides 2006;27 (14):3115-3121.
13. Machida M, Takechi K, Sato H, Chung SJ, Kuroiwa H, Takio S. Genes for the peptidoglycan synthesis pathway are essential for chloroplast division in moss. Proc Nat! Acad Sci USA 2006;103 (4):6753-6758.
14. Kim DH, Lees WJ, Kempsell KE, Lane WS, Duncan K, Walsh ST. Characterization of a Cys115 to Asp substitution in the cell wall biosynthetic enzyme UDP-G1cNAc enolpyruvyl transferase (MurA) that confers resistance to inactivation by the antibiotic fosfomycin, Biochemistry 1996;35:4923-4928.
15. Rogers HJ, Perkins HR, Ward JB. Biosynthesis of peptidoglycan, In:H.J. Rogers (Eds.) Microbial Cell Walls and Membranes, London:Chapman & Hall 1980; 30:239-297.
16. Ahmed El, Francois Sanschagrin, Roger C. Structure and function of the Mur enzymes: development of novel inhibitors, Molecular Microbiology 2003; 47 (1):1-12.
17.张悦,宋晓玲,黄健.微生物多糖结构与免疫活性的关系.动物医学进展2005；26(8)： 10-12.
18. Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of Molecular Biology 1986; 189:113-130.
19. Rosenberg AH, Lade BN, Chui DS. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 1987; 56:125-135.
20. Studier FW, Moffatt BA.Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.Journal of Molecular Biology 1986;189:113-130.
21. Rosenberg AH, Lade BN, Chui DS. Vectors for selective expression of cloned DNAs by T7 RNA polymerase.Gene 1992; 56:125-135.
22. Ventura S. Sequence determinants of protein aggregation:tools to increase protein solubility. Microbial Cell Factories 2005; 4(1):11-19.
23. Carrio MM, Cubarsi R, Villaverde A. Fine architecture of bacterial inclusion bodies.FEBS Letter 2000; 471(1):7-11.
24. Carrio MM, Villaverde A. Construction and deconstruction of bacterial inclusion bodies.Journal of Biotechnology 2002; 96(1):3-12.
25. Schmitt J, Hess H. Affinity purification of histidine-tagged.Molecular Biology Report 1993; 18(3):223-230.
26.高红伟.α2半乳糖营酶活性测定方法的研究.军事医学科学院院刊2006；30(6)：559.
2. 陈国胜,谷欣,张想竹.细菌肽聚糖及其应用.安阳工学院学报2007;6：36-38.
3. 和致中.细菌细胞壁肽聚糖的分类.微生物学通报1992；19(3)：174-179.
4. Green DW. The bacterial cell wall as a source of antibacterial target. Expert Opin.Ther.Tar 2002; 6:1-19.
5. Walsh CT. Enzymes in the D-alanine branch of bacterial cell wall peptidoglycan assembly. J.Biol.Chem 1989; 264:2393-2396.
6. Mao Y, Zhiwen Zhang. Molecular cloning and expression analysis of peptidoglycan recognition protein Ⅱ gene of the large yellow croaker.J.Biotec 2008; 7:1278-1283.
7. Yukari F, Yasuko K. Synthesis of crosslinked peptidoglycan fragments for investigation of their immunobiological functions.Tetrahedron Letters 2009; 4:13-18.
8. Koch AL. Bacterial wall as target for attack:past, present,an future research.Clin.Microbiol Rev 2003;16:673-687.
9. Jenny BS, Brian G. An amino acid substitution that blocks the deacylation step in the enzyme mechanism of penicillin-binding protein 5 of Escherichia coli. Federation of European Biochemical Societies.19841; 25-31.
10. Clifton E, Dean C, Michael R. Targeting the Formation of the Cell Wall Core of M. tuberculosis. Infectious Disorders-Drug Targets 2007; 7:182-202.
II. Ahmed E, Francois S, Roger C. Structure and function of the Mur enzymes: development of novel inhibitors.MolecularMicrobiology 2003; 47(1):1-12.
12. Eschenburg S, Schonbrunn E. Comparative Xray analysis of the un-liganded fosfomycin-target murA.Proteins 2000; 40:290-298.
13. Marquardt JL, Brown ED, Walsh C., Anderson KS. Isolation and structural elucidation of tetrahedral intermediate in the UDP-N-acetylglucosamine enolpyruvoyl transferase enzymatic pathway. J Am Chem Soc 1993; 115:10398.
14. Skarzynski T, Kim DH, Lees WJ, Walsh CT, Duncan K. Stereochemical course of enzymatic enolpyruvyl transfer and catalytic conformation of the active site revealed by the crystal structure of the fluorinated analogue of the reaction tetrahedral intermediate bound to the active site of the C115A mutant of MurA.Biochemistry 1998; 37:2572-2577.
15. Brown ED, Marquardt JL, Lee JP, Walsh CT, Anderson KS. Detection and characterization of a phospholactoyl-enzyme adduct in the reaction catalyzed by UDP-N-acetylglucosamine enolpyruvoyl transferase, MurZ. Biochemistry 1994; 33:10638-10645.
16. Kim DH, Lees WJ, Kempsell KE, Lane WS, Duncan K, Walsh CT. Characterization of a Cys 115 to Asp substitution in the Escherichia coli cell wall biosynthetic enzyme UDP-G1cNAc enolpyruvyl transferase (MurA) that confers resistance to inactivation by the antibiotic fosfomycin. Biochemistry 1996; 35: 4923-4928.
17. Ramilo C, Appleyard RJ, Wanke C, Krekel F, Amrhein N, Evans JN. Detection of the covalent intermediate of UDP-N-acetylglucosamine enolpyruvyl transferase by solution-state and time-resolved solid-state NMR spectroscopy. Biochemistry 1994; 33:15071-15079.
18. Schonbrunn E, Svergun DI, Amrhein N, Koch MH.Studies on the conformational changes in the bacterial cell wall biosynthetic enzyme UDP-N-acetylglucosamine enolpyruvyltransferase (MurA). Eur J Biochem 1998; 253:406-412.
19. Samland AK, Amrhein N, Macheroux P. Lysine 22 in UDP-N-acetylglucosamine enolpyruvyl transferase from Enterobacter cloacae is crucial for enzymatic activity and the formation of covalent adducts with the substrate phosphor-enolpyruvate and the antibiotic fosfomycin.Biochemistry 1999;38:13162-13169.
20. Du W, Brown JR, Sylvester DR, Huang J, Chalker AF. Two active forms of UDP-Nacetylglucosamine enolpyruvyl transferase in grampositive bacteria. J Bacteriol 2000; 182:4146-4152.
21. Nicolle LE. Urinary tract infection:traditional pharmacologic therapies. Am J Med 2002; 113 (Suppl.1A):35-44.
22. Marquardt JL, Brown ED, Lane WS, Haley TM,lchikawa Y, Wong CH, Walsh CT. Kinetics,stoichiometry, and identification of the reactive thiolate in the inactivation of UDP-G1cNAc enolpyruvoyl transferase by the antibiotic fosfomycin. Biochemistry 1994; 33:10646-10651.
23. Kahan FM, Kahan JS, Cassidy PJ, Kropp H.The mechanism of action of fosfomycin (phosphonomycin).Ann NY Acad Sci 1974;235:364-386.
24. Marquardt J., Siegele DA, Kolter R, Walsh CT.Cloning and sequencing of Escherichia coli murZ and purification of its product, a UDP-N- acetylglucosamine enolpyruvyl transferase. J Bacteriol 1992; 174:5748-5752.
25. O'Hara K. Two different types of fosfomycin resistance in clinical isolates of Klebsiella pneumoniae. FEMS Microbiol Lett 1993; 114:9-16.
26. Arca P, Reguera G, Hardisson C. Plasmidencoded fosfomycin resistance in bacteria isolated from the urinary tract in a multicentre survey. J Antimicrob Chemother 1997; 40:393-399.
27. Horii T, Kimura T, Sato K, Shibayama K, Ohta M.Emergence of fosfomycin-resistant isolates of Shiga-like toxin-producing Escherichia coli 026. Antimicrob Agents Chemother 1997; 43:789-793.
28. Baum EZ, Montenegro DA, Licata L, Turchi I, Webb GC, Foleno BD, Bush K. Identification and characterization of new inhibitors of the Escherichia coli MurA enzyme. Antimicrob Agents Chemother 2001; 45:3182-3188.
29. Schonbrunn E, Eschenburg S, Krekel F, Luger K, Amrhein N. Role of the loop containing residue 115 in the induced-fit mechanism of the bacterial cell wall biosynthetic enzyme MurA. Biochemistry 2000; 39:2164-2173.
30. Schonbrunn E, Svergun DI, Amrhein N, Koch MH.Studies on the conformational changes in the bacterial cell wall biosynthetic enzyme UDP-N-acetylglucosamine enolpyruvyltransferase (MurA). Eur J Biochem 1998; 253:406-412.
31. De Smet KA, Kempsell KE, Gallagher A, Duncan K, Young, DB. Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis. Microbiology 1999;145:3177-3184
32. Jose'Molina-Lo'pez, Franc,ois Sanschagrin, Roger C. Levesque. A peptide inhibitor of MurA UDP-N-acetylglucosamine enolpyruvyl transferase:The first committed step in peptidoglycan biosynthesis. Peptides 2006; 27:3115-3121.
1. World Health Organization. Global tuberculosis control:Surveillance, Planning, Financing. WHO Report 2005. Geneva, Switzerland, WHO/HTM/TB/2005.349.
2. Murray CJ, Jopez AD.Global mortality, disability, and the contribution of risk factors:Global Burden of Disease Survey.Lancet 1997; 349(9063):1436-1442.
3. Dye C, Scheele S,Dolin P.Global burden of tuberculosis estimated incidence, prevalence and mortality by country.JAMA 1999; 282(7):677-686.
4. Susan ED, Richard EC. From magic bullets back to the Magic Mountain:the rise of extensively drug-resistant tuberculosis. Nature Medicine.2007; 13(3):295-8.
5. Raviglione MC, Smith IM. XDR Tuberculosis-Implications for Global Public Health. N. Engl. J. Med.2007; 356:656-659.
6. Wise J. Southern Africa is moving swiftly to combat the threat of XDR-TB. Bull World Health Organ.2006; 84(12):924-5.
7. Brennan PJ, Nikaido H. The envelope of mycobacteria. Annual Reviews of Biochemistry 1995; 64:29-63.
8. Crick DC, Brennan PJ, McNeil MR. The cell wall of Mycobacterium tuberculosis. In Tuberculosis (2 nd Edition) 2004; 115-134.
9. 赵乃昕,高屹.应重视肽聚糖结构在细菌分类中的意义-从某些教科书的内容欠妥论述谈起.微生物学通报2004；31(3)：174-175.
10. Ahmed El, Francois Sanschagrin, Roger C. Structure and function of the Mur enzymes: development of novel inhibitors, Molecular Microbiology 2003; 47 (1):1-12.
11. Gordon E, Flouret B, Chantalat L, Heijenoort J, Mengin-Lecreulx D, Dideberg O. Crystal structure of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase from Escherichia coli,J Biol Chem 2001;276 (10):10999-11006.
12. Jose'Molina-Lo'pez, Francois Sanschagrin, Roger C. A peptide inhibitor of MurA UDP-N-acetylglucosamine enolpyruvyl transferase:The first committed step in peptidoglycan biosynthesis, Peptides 2006;27 (14):3115-3121.
13. Machida M, Takechi K, Sato H, Chung SJ, Kuroiwa H, Takio S. Genes for the peptidoglycan synthesis pathway are essential for chloroplast division in moss. Proc Nat! Acad Sci USA 2006;103 (4):6753-6758.
14. Kim DH, Lees WJ, Kempsell KE, Lane WS, Duncan K, Walsh ST. Characterization of a Cys115 to Asp substitution in the cell wall biosynthetic enzyme UDP-G1cNAc enolpyruvyl transferase (MurA) that confers resistance to inactivation by the antibiotic fosfomycin, Biochemistry 1996;35:4923-4928.
15. Rogers HJ, Perkins HR, Ward JB. Biosynthesis of peptidoglycan, In:H.J. Rogers (Eds.) Microbial Cell Walls and Membranes, London:Chapman & Hall 1980; 30:239-297.
16. Ahmed El, Francois Sanschagrin, Roger C. Structure and function of the Mur enzymes: development of novel inhibitors, Molecular Microbiology 2003; 47 (1):1-12.
17.张悦,宋晓玲,黄健.微生物多糖结构与免疫活性的关系.动物医学进展2005；26(8)： 10-12.
18. Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of Molecular Biology 1986; 189:113-130.
19. Rosenberg AH, Lade BN, Chui DS. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 1987; 56:125-135.
20. Studier FW, Moffatt BA.Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.Journal of Molecular Biology 1986;189:113-130.
21. Rosenberg AH, Lade BN, Chui DS. Vectors for selective expression of cloned DNAs by T7 RNA polymerase.Gene 1992; 56:125-135.
22. Ventura S. Sequence determinants of protein aggregation:tools to increase protein solubility. Microbial Cell Factories 2005; 4(1):11-19.
23. Carrio MM, Cubarsi R, Villaverde A. Fine architecture of bacterial inclusion bodies.FEBS Letter 2000; 471(1):7-11.
24. Carrio MM, Villaverde A. Construction and deconstruction of bacterial inclusion bodies.Journal of Biotechnology 2002; 96(1):3-12.
25. Schmitt J, Hess H. Affinity purification of histidine-tagged.Molecular Biology Report 1993; 18(3):223-230.
26.高红伟.α2半乳糖营酶活性测定方法的研究.军事医学科学院院刊2006；30(6)：559.
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